Poly(ethylene glycol) (PEG) is used widely to mediate cell-cell fusion in the production of somatic cell hybrids and in the fusion injection of macromolecules into cultured cells from erythrocytes or liposomes. However, little is known about the mechanism by which PEG induces fusion of cell membranes. The proposed research will continue to examine in molecular detail the influence of PEG on phospholipid vesicles as a model for events in more complex biological membranes. The primary is to elucidate the mechanism of PEG-mediated membrane fusion. The information obtained will be useful for advancing PEG- mediated cell fusion technologies as well as for providing clues to the molecular mechanism of in vivo cell fusion processes such as endocytosis, exocytotic excretion, protein sorting and transport, and viral budding and infection. Work of the past grant period led to a hypothesis for the mechanism of PEG-mediated fusion: formation of fusion intermediates occurs when bilayers containing fluctuational lipid packing defects in the glycerol backbone region are brought into molecular contact by the dehydrating influence of PEG. The resulting probability of fusion is proportional to both the probability of critical inter-bilayer approach and to the probability of packing disruption in either the inner or outer leaflet of the bilayer. The proposed experiments are organized into six aims that will test and then apply this hypothesis to increasingly more complex membranes including ones treated with a viral fusion peptide and cultured cell membranes: 1] test several bilayer components and bilayer conditions for fusogenicity; 2] elaborate the bilayer structural characteristics associated with fusion; 3] test for a critical bilayer structure that seems to correlate with fusion; 4] using several spectroscopic approaches, test whether there exists a common perturbation in bilayer packing associated with PEG-mediated fusion of different model membranes; 5] examine how a very small quantity of the N-terminal "fusion peptide" of influenza virus hemagglutinin induces fusion of membranes aggregated by low concentrations of PEG; and 6] determine whether bilayer perturbants that act as fusogens in model membranes will increase the efficiency of PEG-mediated fusion of cultured cells.